|Masler, Edward - Pete|
Submitted to: Archives of Insect Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/18/1996
Publication Date: N/A
Citation: Interpretive Summary: EDNH is a very important peptide hormone that is necessary for reproduction in flies. Without this hormone flies will not develop eggs and will remain sterile. EDNH causes ovaries to produce ecdysone. Ecdysone is required for molting and yolk production. Interfering with ecdysone production would provide a very specific, environmentally friendly means of controlling pest insects. We have obtained highly purified EDNH which will provide material for the final purification and can be used in assays to find materials that will inhibit ecdysone production.
Technical Abstract: Highly purified Musca egg development neurosecretory hormone (EDNH) was obtained by using several extraction procedures followed by four chromatography steps. House fly heads were extracted with an acid-ethanol-urea extraction method (Wheelock et al., 1991, J. Chromatography 542, 508-514). The extract was desalted on a Sephadex G25F column and fractionated on a Sephadex G50F column. Active material from the G50 column was loaded on to a semi-preparative C18 reverse phase HPLC column. An acetonitrile gradient in 0.1% trifluroacetic acid in water was employed and ecdysteroidogenic activity was found in a fraction that eluted with 35% acetonitrile. This fraction was then chromatographed on a I-125 size exclusion HPLC column. Ecdysteroidogenic activity was found in a fraction with a molecular weight of 7.5 kDa The extraction steps resulted in a 42,000 fold purification of EDNH. Dose response curves were constructed for active fractions from the G-50 column and the C18 semi-preparative column. Material from the Sephadex column had the highest ecdysteroidogenic activity in vitro at a concentration of 250 ng/ul. The material from the C18 column was most active in vitro at a concentration of 40 ng/ul and stimulated egg development in vivo at a concentration of 1.5 ug/fly. The EDNH in vitro and in vivo yielded tachyphylactic dose-response curves.